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Parasitology ResearchFounded as Zeitschrift fürParasitenkunde ISSN 0932-0113 Parasitol ResDOI 10.1007/s00436-016-5094-2

Human immune response to salivaryproteins of wild-caught Phlebotomuspapatasi

Rami M. Mukbel, RehabH. Khasharmeh, Nawal S. Hijjawi,Mohammed S. Khalifeh, Ma’monM. Hatmal & Mary Ann McDowell

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ORIGINAL PAPER

Human immune response to salivary proteins of wild-caughtPhlebotomus papatasi

Rami M. Mukbel1 & Rehab H. Khasharmeh2& Nawal S. Hijjawi2 &

Mohammed S. Khalifeh1& Ma’mon M. Hatmal2 & Mary Ann McDowell3

Received: 10 December 2015 /Accepted: 26 April 2016# Springer-Verlag Berlin Heidelberg 2016

Abstract Phlebotomine sand flies are the known vectors ofLeishmania parasites. New approaches in vaccination againstLeishmania have investigated the possibility of integratingPhlebotomus papatasi salivary proteins to enhance the im-mune response and protect against the transmission of theinfection. The aim of the present study was to screen humanimmune responses to wild sand fly saliva and evaluate immu-nogenic salivary proteins. Blood samples were collected fromdonors in control and sand fly infested areas. Antibodies spe-cific for sand fly antigens in donor plasma were probed usingimmunoblotting. In addition, recall proliferation capability ofperipheral blood mononuclear cells (PBMC) was tested aftersand fly salivary homogenates stimulation. The significantimmunogenic salivary proteins (SPs) identified by immuno-blotting were SP28, SP32, and SP36. A specific proliferativeresponse of PBMC after stimulation with sand fly salivaryhomogenates was evident in donors that have antibody re-sponses against sand fly salivary proteins. Individuals withantibody recognition to a higher number of salivary proteins(i.e., 3 or more SP bands) showed lower PBMC proliferative

responses after in vitro stimulation with salivary gland ho-mogenates (SGH) only in the sand fly infested, leishmaniasisfree area. Interestingly, the presence of a humoral immuneresponse to many SP antigens inversely correlates with astrong cell-mediated immune response (CMI). It was also no-ticed that some other heavily expressed antigens, in sand flysalivary homogenate, lack or have weak humoral immunereactivity in exposed individuals. Therefore, considering theseantigens alone as CMI activators, without including theimmunodominant humoral immune response proteins, needsfuture investigation.

Keywords Sand fly . Salivary proteins . Proliferation .

Leishmania .Phlebotomus papatasi . Leishmaniamajor

Introduction

Leishmaniasis is a vector-borne parasitic disease with a globaldistribution that reaches approximately two million new casesreported annually (WHO 2015). Leishmaniasis is transmittedby the bites of female Phlebotomine sand flies (Bates 2007;Lane et al. 1993). The clinical manifestations of leishmaniasisare quite diverse depending on the infecting Leishmania spe-cies, the geographic location, and the immune status of thehost (Desjeux 1996; Kebaier et al. 2006). There are threeprimary clinical forms of leishmaniasis depending on the dis-ease manifestation, organ involved, and the geographical dis-tribution which are categorized as visceral leishmaniasis (VL),cutaneous leishmaniasis (CL), and muco-cutaneous leishman-iasis (MCL). The disease ranges from self-healing CL to se-vere systemic disease and is generally fatal if left untreated asin case of VL (Herwaldt 1999; Reithinger et al. 2007).Leishmania spp. have a digenetic life-cycle, alternating be-tween extra-cellular promastigotes in Phlebotomine sand flies

Rami M. Mukbel and Rehab H. Khasharmeh contributed equally to thiswork.

* Rami M. Mukbelrmmukbel@just.edu.jo

1 Department of Basic Veterinary Medical Science, Faculty ofVeterinary Medicine, Jordan University of Science and Technology,P.O. Box 3030, Irbid 22110, Jordan

2 Department of Medical Laboratory Sciences, Faculty of AlliedHealth Sciences, The Hashemite University, PO Box 150459,Zarqa 13115, Jordan

3 Eck Institute for Global Health, Department of Biological Sciences,University of Notre Dame, Notre Dame, IN 46556, USA

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(insect-stage) to obligate intracellular amastigotes within cellsof the vertebrate host (mammalian-stage) (Cecilio et al. 2014).

Zoonotic cutaneous leishmaniasis (ZCL) is an endemicdisease in Jordan, mostly caused by L. major with a reportedprevalence rate at 313/105 persons between year 2004 and2005 (Mosleh et al. 2008). The disease is primarily maintainedby the reservoir host Psammomys obesus and the sand flyvector Phlebotomus papatasi (Janini et al. 1995; Kamhawiet al. 1993). Inhabitants of areas endemic for leishmaniasisexhibit attenuated Leishmania infections and are less likelyto contract the disease as compared to newcomers (Andradeet al. 2007). This observation has been attributed to the repeat-ed exposure of individuals to non-infected sand fly bites that isthought to induce a protective immunity against Leishmaniainfection (Kamhawi 2000). It is important to address that thisprotection induction was achieved from pre-immunizationwith salivary proteins of P. papatasi collected from long-term colonized P. papatasi (Valenzuela et al. 2001a, b). Onthe other hand, natural and frequent exposures in endemicareas to non-infective bites of P. papatasi do not result insimilar disease protection. Therefore, it is clear that wild-caught and/or recently colonized P. papatasi immunologicalbehavior is different from that long-term colonized P. papatasi(Ahmed et al. 2010; Ben Hadj Ahmed et al. 2010, 2011).

Sand fly females release approximately 10 to 1000Leishmania parasites along with saliva during the blood-feeding process (Kimblin et al. 2008; Rogers et al. 2004;Titus and Ribeiro 1988). Sand fly saliva is composed of potentpharmacologically active molecules that act as anticoagulants,anti-platelets, and vasodilators and have immunomodulatoryand anti-inflammatory activities (Abdeladhim et al. 2014;Charlab et al. 1999; Kamhawi et al. 2000). Sand fly salivacomponents are important in facilitating the blood-feedingprocess (Ribeiro and Francischetti 2003; Rohousova andVolf 2006). For example, P. papatasi secretes adenosine andadenosine monophosphate (AMP) during a blood meal(Ribeiro et al. 1999). Both molecules act as vasodilators andexhibit anti-platelet aggregation properties (Dionisotti et al.1992). Furthermore, P. papatasi saliva also contains a signif-icant level of endogenous protein phosphatase 1 (PP-1) andPP-2A like activity (Desjeux 2004; Katz et al. 2000).Moreover, an anti-platelet aggregation enzyme known as ap-yrase (SP36) has also been identified in P. papatasi (Chatelainet al. 1992; Valenzuela et al. 2001b).

Several studies have been conducted to characterize themain components of the saliva and their role in the immuneresponse initiation. It was reported that P. papatasi salivaryproteins elicit antibody responses in mice (Rohousova et al.2005; Valenzuela et al. 2001a, b). These saliva immunogensconsisted of 12 major protein bands ranging in size from 10 to100 kDa (Valenzuela et al. 2001a). It is generally thought thatthe enhancement of Leishmania infections by sand fly salivais due to its non-specific immune-modulatory activities.

Conversely, the delayed-type hypersensitivity (DTH) elicitedby the repeated exposure to sand fly bites or salivary glandhomogenates (SGH) inhibits Leishmania infection (Belkaidet al. 1998). Vaccination with SGH, or pre-exposure to unin-fected sand fly bites, modulates the cytokine milieu at the siteof parasite delivery toward a T-helper 1 (Th1) response that isnecessary to generate cell-mediated immune responses againstLeishmania parasites. The principle objective in the currentwork was to screen for saliva proteins from wild-caughtP. papatasi that are immunogenic to humans in a naturalsetting.

Materials and methods

Insects collection and salivary gland homogenates (SGH)preparation

Adult sand flies were collected during the sand flies activityseason once weekly from June to September during 2011 and2012 (Schlein and Muller 2010) (Table 1). In addition, mos-quitoes (Culex sp.) were also collected for the purpose ofcomparison with the collected sand flies salivary proteins.Collections were carried out using CDC light traps (John W.Hock Company, USA) in Alkhaldeih (Mafraq) and Swaymeh(Dead Sea) Jordan; both areas are known to harbor high andcomparable abundance of P. papatasi (Kamhawi et al. 1995).Traps were placed near potential resting and breeding sitesaround houses and near reservoir host burrows with more than90 % collected from the resting sites. Generally, traps wereplaced at sunset and collected before the following sunrise.Collected flies were transferred in the trap net bags whichwere kept humidified in plastic bags and transferred back tothe laboratory where the sand flies were removed andidentified.

Table 1 Number of salivary glands collected per time of the year permonth

Collection time Number of salivary glands collected per area

Year Month Swaymeh Mafraq

2010 August 55

September 42

2011 June 3 19

July 5 21

August 279

September 39

2012 August 366

September 50

784 95

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Collected flies were immersed in 1 % detergent soap solu-tion for killing and body hair removal and then washed threetimes in tap water. Flies were then transferred to containerwith sterile (1X) phosphate buffered saline (PBS, Lonza,USA) and kept cool on ice until dissected using an Olympussz61 zoom stereomicroscope. The flies were identified basedon specific morphological features according to the keys de-veloped by Lewis (1978).

The spermatheca in female sand flies was used for speciesidentification. Briefly, the terminal abdominal segment wasseparated and placed in a drop of (1X) PBS on a clean micro-scope slide and pressed gently with the cover glass to flatten itto facilitate species identification by light microscopy (MoticBA 200 Trinocular Biological Microscope, Australia). Afterpositive identification of female P. papatasi flies which weremore than 95 % of trapped sand flies, the salivary glands wereextracted on the same day of trapping by pulling the headgently from the body using forceps and needles; the visualizedglands were carefully scooped into a storage tube using sy-ringe head (Titus and Ribeiro 1988; Volf et al. 2000). In orderto cover all the variations between time and sites of collection(Ramalho-Ortigao et al. 2015; Volf et al. 2000), salivaryglands were pooled in groups of 20 in a volume of 20 μL(1X) PBS in 1.5 mL micro-centrifuge tubes and stored at−70 °C. Immediately before use, salivary gland homogenates(SGHs) were prepared by exposing the glands to three freeze-thaw cycles then sonicated (Bransonic 5510 E-MT, Mexico)and centrifuged (10,000 RCF for 2 min) (Abdeladhim et al.2011). SGH used for peripheral blood mononuclear cells(PBMC) stimulation and proliferation assay were filter steril-ized through 0.22 μm ultra-free centrifugal filter units(Millipore).

Blood samples

In the period between July 2012 and December 2012, a totalof 114 blood samples from donors living in three differentlocations, Swaymeh, Amman, and Mafraq, were collected.The selected sites were chosen according to the presence orabsence of both sand flies and Leishmania parasites as sum-marized in the Table 2. It is important to note that the selectedareas are in close proximity and traveling for people amongthese areas is expected. In addition, it was observed that the

abundance of trapped P. papatasi was comparable at the twoexperimental areas (MF and SW). Samples from peripheralblood (9 mL) from the vein of volunteers’ were collected invacutainer tubes with acid citrate dextrose solution A (ACD-A, Greiner Bio-One, Austria). Blood plasma was separated bycentrifugation of blood at 1500 RCF for 10 min at room tem-perature before being transferred to cryo-tubes and then frozenat −70 °C. The donors were asked to sign a consent formregarding the work conducted and individual informationwas filled for each donor separately. Data for each donor werecollected in a questionnaire that was filled during interview.All personal information has been kept secret. All donors par-ticipating in this study were healthy and above 18 years of age.

Peripheral blood mononuclear cells isolation

PBMCs were separated using Ficoll-hypaque density gradientcentrifugation. Briefly, fresh blood samples were diluted (afterplasma separation) twofold with sterile (1X) PBS and slowlylaid over the Lymphocyte Separation Medium (LSM1.077,Lonza, USA). After centrifugation for 30 min at 900 RCF,the mononuclear cell layer was collected into a separate15 mL tubes and washed by adding Hanks balanced salt so-lution (HBSS, EuroClone, Italy) before centrifugation for10 min at 400 RCF. RBCs were subsequently lysed with lysisbuffer (Promega, USA) for 5 min on ice. PBMCs were thenwashed two times with tissue culture medium (RPMI-1640,EuroClone, Italy) by centrifuging for 10 min at 250 RCF.Finally, the pellet was re-suspended in 1 mL completeRPMI-1640 supplemented with 10 % heat inactivated fetalbovine serum (FBS), 2 mM L-glutamine, and 1 % penicillin/streptomycin (100 U/mL penicillin and 100 μg/mL strepto-mycin) (all supplements were obtained from EuroClone,Italy). Viable cells were counted using trypan blue (Sigma-Aldrich, Germany) exclusion test by hemocytometer chamber(Marienfeld, Germany) (Chang et al. 2007; Freshney 1987;Gantt et al. 2001).

Cell proliferation

Cell proliferation has been assessed using a non-radioactive 5-bromo-deoxyuridine (BrdU) incorporation assay. For eachsample, 12 wells with 1×105 PBMC per well were plated in

Table 2 Selected residential sitesof human peripheral blood donorsfrom Jordan

Selected sites Phlebotomus papatasi Leishmania majortransmission

Abbreviation Number of donors(male/female)

Amman Absent Absenta AM 38 (21/17)

Mafraq Present Absent MF 38 (19/19)

Swaymeh Present Presenta SW 38 (9/29)

Total 114 (49/65)

a Present and absent indicate known cases of active L. major transmission in the region

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a U-shape 96 well tissue culture plate setup. SGH was addedat a concentration of 0.5 salivary gland per mL and phytohe-magglutinin (PHA; 10 μg/mL) (Remel, UK) was used as apositive proliferation control (Geraci et al. 2014). For eachdonor, wells without any stimulation were used as a negativecontrol. Total volumes were adjusted to 200 μL per well andplates were incubated in a 5 % CO2 humidified incubator at37 °C for 5 days.

At day 4, BrdU was added to the wells then further incu-bated for 18 h. The proliferation was then measured usingBrdU Enzyme Linked Immunosorbent Assay (ELISA) kit(BrdU [colorimetric}, Roche, Germany) according to manu-facturer’s instruction. All solutions, buffers, and media used intissue culturing were sterilized using a 0.22 μm Vacuum fil-tration unit (JET BIOFIL, USA) and kept at 4 °C for furtheruse. Moreover, cells were treated under sterile conditions inendotoxin free environment and exclusively under ESCOclass II Biological Safety Cabinet). Proliferation results wereexpressed as stimulation index (SI) which represents the meanof antigen-stimulated cultures/mean of un-stimulated culturesin all tested individuals.

SDS-PAGE

Pooled proteins of wild-caught sand fly salivary glands as wellas mosquitoes (Culex sp.) salivary proteins were separatedusing the sodium dodecyl sulfate polyacrylamide gel electro-phoresis (SDS-PAGE) technique to characterize the salivaryproteins of P. papatasi collected from all selected study areas(Ramalho-Ortigao et al. 2015). Briefly, Tris-glycine gels(12 %) with a thickness of 1 mm were prepared usingModular Mini Vertical Gel and Blotting System (MajorScience, Taiwan) according to manufacturer’s protocol.

Before use, salivary glands were subjected to three freeze-thaw cycles then sonicated for 1 min and centrifuged at 8000RCF for 2 min. An equivalent of 20 glands was used per gel.Furthermore, the glands were lysed in 16 μL 2X SDS samplebuffer (0.125 M Tris-Cl, pH 6.8, 4 % SDS, 20 % glycerol,10 % 2-mercaptoethanol, 0.25 % bromophenol blue), 4 μL 2-mercaptoethanol and 50 μL PBS, and incubated at 85 °C for10 min. The mixture was then cooled down immediately onice then centrifuged at 13,000 RCF for 3 min. Finally, the twolanes of the gel were loaded, one with salivary gland lysateand the other with 2.5 μL ladder’s (BlueRAY Pre-stainedProtein Ladder markers, GeneDirex, USA). Electrophoresiswas carried out at 125 V 40 mA for 100 min using Tris-Glycine running buffer with SDS (0.025 M Tris, 0.192 MGlycine and 1 % SDS).

Staining

Gels were placed in a fixation solution (50 % methanol, 10 %acetic acid) overnight then stained for 4 h in 1 % Coomassie

Brilliant Blue R-250 (Sigma-Aldrich, Germany). The gel wasde-stained overnight in de-staining solution (5 % methanol,7.5 % acetic acid) and stored in 7 % acetic acid. The gel imagewas taken using the doc Universal hood ΙΙ (Bio-Rad, USA).

Immunobloting

To identify the immune reactive salivary proteins recognizedby sand fly bitten individuals, western blot analysis ofP. papatasi SGH was performed (Barral et al. 2000; Geraciet al. 2014). Briefly, separated proteins were transferred onto anitrocellulose membrane using the Modular Mini Electro BlotSystem (Major Science, Taiwan). After protein transfer, thenitrocellulose membrane was blocked for 1 h in 0.1 %Tween/Tris-buffered saline (100 mM Tris HCL, pH 7.5,0.9 % NaCl) (TBS-T) with 5 % non-fat dried milk.Membranes were then placed in a Mini-protean II multi-screen apparatus (Bio-Rad, USA) to allow the screening ofplasma samples. Plasma samples were diluted 1:100 inblocking buffer (2 % non-fat milk-TBS-T) and applied to theblot overnight at 4 °C on a rotating platform.Membranes werethen removed from the cast and washed 3 times with TBS-T(15 min for each wash). Goat anti-human immunoglobulinconjugated to alkaline phosphatase (goat anti-human IgGF(ab’)2 Alkaline Phosphatase, AbD Serotec, UK) secondaryantibodies were used at a 1:5000 dilution and applied to blotsfor 1 h at room temperature. Recognized proteins were visu-alized using the colorimetric substrate BCIP/NBT (BCIP/NBT Color Development Substrate, Promega, USA). Allchemicals for SDS-PAGE and western blot assays were ob-tained from Bio Basic Canada Inc. The blots were scannedusing computer scanner for documentation.

Statistical analysis

The proliferation assay results and the comparison betweenthe different areas were performed using two-way analysisof variance (ANOVA) test at 95 % confidence interval formean. The significant differences among means were testedusing Student’s t test. For all tests and comparisons, the dif-ference was considered significant at P<0.05.

Ethics statement

This study was approved by the ethic committee of theInstitutional Review Board (JUST-IRB) at Jordan Universityof Science and Technology and performed according toDeclaration of Helsinki principles. The donors were asked toread and sign a consent form regarding the work conductedand individual information was filled for each donor separate-ly. The consent form informed the participants of any potentialrisks of the study.

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Results

Humoral antibody response

SDS-PAGE of separated SGH of P. papatasi were stained byCoomassie Brilliant Blue which revealed approximately 11heavily expressed salivary proteins (SP14, SP15, SP28,SP30, SP32, SP36, SP42, SP44, SP46, SP52, and SP64) andone modestly expressed protein, SP12 (Fig. 1). Plasma from114 donors was tested against PAGE-separated SGH fromwild female P. papatasi by western immunoblot. Antibodiesof donor plasma displayed differential specificities for somesalivary proteins, after transfer to western blot (Fig. 2). Therewere individuals that had antibody responses to some specificbands (SP28, SP32, SP36, and SP42) while other heavilyexpressed bands for SGH on SDS-PAGE (SP14, Sp15,SP30, SP44, and SP46) showed no humoral immune reactiv-ity on immunoblotting assay (Table 3). Reactivity to the heavi-ly expressed sand fly salivary protein SP64 was observed inapproximately half of donors (54 donor out of 114), from allthree areas. Interestingly, the same donor plasma recognized asimilarly sized protein in mosquitoes (Culex sp.) SGH(Fig. 3). This cross-species reactivity drives the exclusion ofthis antigen (SP64) from further use as a vaccine candidate.

The immunoblotting results were compared between areasinfested with sand flies (Mafraq and Swaymeh) (Table 3).High levels of antibody reactivity for three salivary proteins(SP28, SP32, and SP36) were observed in Mafraq, while inSwaymeh, additional, moderate, antibody reactivity was

observed to another salivary protein (SP42). In general, pro-teins such as SP12, SP30, SP46, and SP52 provoked weakhumoral immune responses in individuals living in areasinfested with sand flies. Although they heavily expressed inSGH, reactivity to SP14, SP15, and SP44 was not detected inany individuals.

Cell-mediated immune response

Among all experimental groups, and regardless of the crudeSGH concentration used to stimulate PBMC, the negativecontrol area had the lowest cell proliferation SI (Fig. 4). Ingeneral, individuals fromMafraq, where sand flies are presentwithout reported leishmaniasis cases, had the highest SI whencompared to other areas. Finally, cell proliferation SI values ofindividuals from Swaymeh, an area endemic with L. major,was significantly higher than SI observed in negative controlarea (Amman) but lower thanMafraq, especially when PBMCstimulated with a lower SGH concentration. It is important tonote that samples from Amman that showed a positive anti-body response in the immunoblotting assay to SGH (19 sam-ples), as well as samples from Mafraq (5 samples) andSwaymeh area (4 samples) that showed no humoral immuneresponse against SGH, were excluded from SI comparison.

We further compared cell proliferation SI in accordancewith the frequency of specific antibody reactivity to differentsalivary P. papatasi proteins in individuals resident in sand flyinfested areas (Fig. 5). Individuals with antibody reactivity to

Fig. 1 Salivary protein profile of Jordanian wild Phlebotomus papatasi.Awhole salivary gland protein homogenate isolated from Jordanian wildPhlebotomus papatasi. Salivary gland proteins were separated using12 % SDS-PAGE gel electrophoresis stained with Coomassie BrilliantBlue R-250. The bands where compared visually with existing salivaryprotein profile of colonized P. papatasi according to Valenzuela et al.(2001a). (Numbers indicate the position of the molecular weight markerin the same gel)

Fig. 2 Immunoblot of reactive Phlebotomus papatasi salivary proteins.Immunoblot reactive bands of donor plasma from the three selected siteswith antibody specificity to Phlebotomus papatasi salivary proteins.Lanes were designated by regional serum donor codes: AM, MF, andSW (see Table 2), positive donor designated as (+ve), and negativecontrol designated as (−ve)

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many antigen panels in the immunoblot assay exhibited lesscell proliferation to SGH in samples collected from Mafraq.This adverse effect was only apparent in samples stimulatedwith 0.5 and 1 gland per mL (Fig. 5a). Regardless of SGHconcentration, PBMCs collected from donors living inLeishmania-endemic area (i.e., SW) had no significant differ-ences in the proliferation index results (Fig. 5b, c). Thus, SWindividuals with antibody reactivity to 3 or more SP antigenson immunoblotting assay had similar proliferation index tothose individuals with antibody reactivity to two or less bands.

Discussion

The goal of the present study was to identify sand fly salivaryproteins that activate cellular and humoral immune responsesin volunteers naturally exposed to frequent bites by the wildsand flies (P. papatasi). The analysis of specific circulatingantibodies to whole sand fly salivary gland homogenates, aswell as the cellular response elicited by the presence of mem-ory lymphocytes, was investigated. Several previous studiessuggested that exposure of mice to sand fly bites results instrong cellular and humoral immunity specific to salivary

gland proteins (Rohousova and Volf 2006; Silva et al. 2005).Analysis of cellular immunity against sand fly saliva in vol-unteers exposed to the bites of P. papatasi is limited(Abdeladhim et al. 2011; Geraci et al. 2014; Marzouki et al.2011). In comparison to other studies that used salivary pro-teins obtained from colony raised sand flies, the current workspecifically assessed the proliferative immune response to-ward the salivary gland homogenates collected from wild-caught sand flies (i.e., P. papatasi) with the presence of spe-cific antibodies in people naturally exposed to sand fly bites,in leishmania-endemic and leishmania free, sand fly infestedareas.

A higher cellular proliferation rate in response to SGHstimulation was identified among donors from sand flyinfested areas compared to donors from the negative controlarea. Cellular proliferation was inversely correlated with anincreased antibody response activity in individuals that wereresident in sand fly infested areas but free of leishmaniasis(MF). The most interesting finding in the current work is thatindividuals exhibiting humoral immunity to a higher numberof salivary proteins had a lower ability to mount a strong cellproliferative response. It was also remarkable that in the leish-maniasis endemic area (SW), individuals do not exhibit a cell

Table 3 Frequency of individuals reacting with antibody response detected by immunoblotting assay against specific salivary Phlebotomus papatasiproteins in Leishmania-endemic (Swaymeh) and non-endemic (Mafraq) areas

SP12 SP14 SP15 SP28 SP30 SP32 SP36 SP42 SP44 SP46 SP52

Mafraq ND ND ND 20 1 30 20 3 ND 1 1

Swaymeh 2 ND ND 21 ND 32 19 8 ND 2 6

Total 2 ND ND 41 1 62 39 11 ND 3 7

ND not detected

a bPhlebotomus

papatasiMosquitos

Phlebotomus

papatasiMosquitos

Fig. 3 Salivary protein profileand immunoblot of Jordanianwild mosquito (Culex sp.). a Awhole salivary gland proteinhomogenate of Jordanian wild P.papatasi and a whole salivarygland protein homogenate ofJordanian wild mosquitoes (Culexsp.) run on the same denaturing12 % SDS-PAGE gel and stainedwith Coomassie Brilliant BlueR-250. b Immunoblot reactivebands of donor plasma from thethree selected sites with antibodyspecificity to mosquitoes andP. papatasi salivary proteins runon the same gel. Lanes aredesignated by regional serumdonor codes: AM, MF, and SW(see Table 2)

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proliferative response regardless of antibody reactivity orSGH concentration. These data may suggest that individualswith a low humoral immune response (possibly earlyrespondents) that reside in sand fly areas free formleishmaniasis have strong cell-mediated immunity responseswhen their immune cells are exposed to sand fly antigens.Thus, the suggestion of tolerance induction with high SGHstimulation seems as an appealing explanation (Rohousova etal. 2011). Therefore, these findingsmight explain the high riskincidence of leishmaniasis in endemic areas even with theexistence of strong humoral immune responses against sandfly salivary proteins (Barral et al. 2000; Gomes et al. 2002;Rohousova et al. 2005). In addition, the current work suggestsa new approach in handling and assessment of immunologicalsand fly salivary proteins. Although the salivary protein typesplay several important roles in disease transmission anddisease pathogenesis, the emphasis on the number ofimmunogenic salivary proteins, regardless of the proteintypes in individuals, need to be considered when evaluatingand determining the immunological behavior against SGHexposure.

Immunodominant protein bands that elicit a humoral hostimmune response could be employed as biomarkers (i.e.,SP32) for human exposure to sand fly bites (Marzouki et al.2015). Moreover, some of proteins with high antibodyreactivity can be used to map a sand fly species spectrum inan attempt to identify vector exposure in a certain area. Inaddition, studies have strongly supported, for example, thepossibility of using P. papatasi saliva antibodies asindicators of vector exposure, a potentially useful parameter

in forecasting the development of Leishmania majorinfections (Abdeladhim et al. 2014; Sacks et al. 1995).Clearly, individuals living in sand fly infested areas exhibitincreased humoral responses to sand fly saliva compared toindividuals residing in uninfested areas. The major humoralimmune reactive salivary proteins can be labeled as SP28,SP32, SP36, and SP42 with the SP32 being theimmunodominant one followed by SP28 and SP36, somewhatconsistent with previous reports (Abdeladhim et al. 2011).Interestingly, the immunodominant antigen was not the samebetween the two sand fly infested sites. The number of indi-viduals responding to SP42 was 3 donors in MF, while thenumber of individuals in SW reached 8.

The close proximity of several proteins on immune blots,such as bands present between the 25 and 35 kDa cluster(Fig. 2), make it difficult to distinguish the exact type of theSP identified by humoral immunity. For example, the closelocation of SP30 and SP32 on the immunoblotting assaymight lead to mis-interpretation of the importance and identityof each of these SP antigens (Marzouki et al. 2012). Therefore,previous studies concluded that presence of a protein on asingle SDS-PAGE does not always mean one protein but rath-er suggested several proteins sharing the same band (Geraciet al. 2014; Marzouki et al. 2012). However, the antibodyreactivity toward the three close bands (i.e., SP28, SP30 andSP32) that was present in one volunteer (data not shown) wasemployed as a guide to approximately identify the antibodyreactivity to each of these bands in other individuals.Accordingly, the band identified with most antibody reactivityin the current workwas labeled as SP32 and not SP30. Despitethe above approach in labeling and defining the salivaryproteins, data analysis in the current work did not give muchweight to each individual band type or size but rather theanalysis was completely based on the number of recognizedbands in each responder. Therefore, regardless of the type ofSP proteins, exposed individuals were divided into lowantibody responders (1 or 2 bands) compared with highresponders (3 or more bands). Accordingly, PBMCproliferation in response to different SGH concentrationswas compared in these two antibody responding groups.

Strikingly, little specificity for SP12, SP30, SP46, andSP52 salivary proteins was observed and no antibodyresponse was reported toward SP14, SP15, and SP44salivary proteins. Thus, this study also hypothesizes that theweak, or the absence of, humoral immune response for someheavily expressed salivary proteins might point out that theseantigens should be further explored for their ability tostimulate cell-mediated immunity. Interestingly, a recent studyindicated that the immune response to a single sand fly sali-vary protein may differ from the response to SGH, and adistinct protein from the same vector can generate a differentimmune response by skewing to a Th1 or Th2 pattern(Oliveira et al. 2008). For example, SP15 which had no

Fig. 4 The index of proliferation in all tested individuals at the threeselected sites. The stimulation index (SI) of peripheral bloodmononuclear cells (PBMC) in response to P. papatasi salivary glandhomogenates at a concentration of 2, 1, and 0.5 salivary gland per mL.PBMC were isolated from volunteer donors; bars are designated byregional donor codes: control area (i.e., Amman (AM)) and sand fliesinfested area (Mafraq (MF) and Swaymeh (SW)). Stimulation indexexpresses the proliferation mean in antigen-stimulated cultures/proliferation mean in un-stimulated culture. Each value represents themean value ± S.E.M. Different letters indicate a significant differencebetween groups (P< 0.05)

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antibody reactivity in the current study was previously dem-onstrated to activate a cell-mediated immune response andrepeatedly reported to enhance protection againstLeishmania challenge in murine hosts (Elnaiem et al. 2005;Oliveira et al. 2008; Valenzuela et al. 2001a) and non-humanprimate models using SP15 from P. duboscqi (Oliveira et al.2015). Of note is that protection against L. major infectionimproved in B cell-deficient mice vaccinated with SP15(Valenzuela et al. 2001a). From this point of view, it wassuggested that the protein PpSP15 from P. papatasi as wellas P. dubosqui saliva would be a better cell-mediated immuneactivator and thus a promising candidate for future vaccinedevelopment (Elnaiem et al. 2005; Lestinova et al. 2015;Oliveira et al. 2008). Similarly, SP44 did not stimulate anyhumoral antibody response in the current study although ithas been previously demonstrated to induce a strong IL-4response in mice (Oliveira et al. 2008). As well, this proteinwas also shown to have an adverse effect on protection ininfected mice (Oliveira et al. 2008). In addition, it is wellestablished that most blood-feeding arthropods that are re-sponsible for transmission of many diseases have immuno-suppressive proteins in their saliva (Titus et al. 2006).Therefore, some antigens with low or no humoral immuno-genic responses reported in the present study such as SP14,SP15, and SP44 might have characteristics worthy of furtherinvestigations to clarify their exact immune-modulatoryeffects.

The differential immune responses toward separate sali-vary proteins suggest that the immune cells in these recruitedindividuals respond to one type of immune arm of defenseover another in response to these proteins. Thus, proteins suchas SP28, SP32, SP36, and SP42 were known to stimulate theantibody response in the majority of exposed donors. Thepresence of SP36 (apyrase) in sand fly SGH might havecaused the suppression of the proliferative responses ofPBMC by increasing the hydrolytic reactions to boost AMPlevels in dendritic cell cultures (Carregaro et al. 2011).Marzouki et al. (2011) found that all positive sera from natu-rally exposed donors strongly reacted with a P. papatasi 30-kDa protein band which correspond to SP30 using mass spec-trometry. Interestingly, later conformational studies by thesame group showed that SP32 salivary protein from

P. papatasi is the immunodominant one (Marzouki et al.2012). Indeed, adenosine has been shown to be present inlarge quantities in P. papatasi salivary glands (Ribeiro et al.1999), and it has been reported to enhance the production ofIL-10 and decrease the production of IL-12 and IFN-γ(Carregaro et al. 2011) However, even in this adenosine pres-ence, significant differences were identified in the prolifera-tion results in the current study (Figs. 4 and 5).

It is important to emphasize that although the selected sitesin Jordan, representing control and experimental regions, areclimatically diverse, they are relatively in close geographicalproximity. In addition, the ease of movement within theseareas might be an important factor that affected the antibodyspecificity profiles of donors against SGH despite the charac-teristics of their primary residence sites. This might explainthat several individuals in the Amman area, which supposedlyhad no previous exposure to sand fly salivary proteins, eliciteda humoral response against several SGH bands.

The significant appearance of SP64, and to some extentSP52 in several volunteers in all experimental groups, couldbe attributed to the possibility of volunteers’ exposure to sal-ivary gland proteins of other non-sand fly hematophagousarthropods such as mosquitoes. Antibodies with specificityto potentially conserved protein domains among the differentinsects (Fig. 3) might explain the arbitrary antibody reactivityappearance in several individuals. This might also suggestcommon conserved protein in blood sucking insects that couldcause cross reactivity in host humoral immune response(Francischetti et al. 2002).

The variations in antibody specificity for different salivaryproteins were clearly obtained by naturally exposed individ-uals to saliva of P. papatasi. The most reactive salivary pro-teins in endemic areas were SP28, SP32, and SP36 with theSP32 being immunodominant for antibody production. Thelow PBMC proliferation response that appeared in individualswith high humoral reactivity suggests the need to explore thespecific cell-mediated immune responses toward other SGHheavily expressed proteins that were lacking humoral immuneresponse stimulation ability alone without including humoralimmunodominant proteins.

References

Abdeladhim M, Ben Ahmed M, Marzouki S, Belhadj Hmida N,Boussoffara T, Belhaj Hamida N, Ben Salah A, Louzir H (2011)Human cellular immune response to the saliva of Phlebotomuspapatasi is mediated by IL-10-producing CD8+ T cells and Th1-polarized CD4+ lymphocytes. PLoSNegl Trop Dis 5:e1345. doi:10.1371/journal.pntd.0001345

AbdeladhimM, Kamhawi S, Valenzuela JG (2014) What’s behind a sandfly bite? The profound effect of sand fly saliva on host hemostasis,

�Fig. 5 The stimulation index in individuals with low and high antibodyreactivity toward different numbers of salivary P. papatasi proteins. Thestimulation index (SI) of peripheral blood mononuclear cells (PBMC) inresponse to P. papatasi salivary gland homogenates at a concentration of0.5 (a), 1 (b), and 2 (c) salivary gland per mL. PBMC were isolated fromvolunteer donors from sand flies infested area (Mafraq (MF) andSwaymeh (SW)). The SI results for the sand flies exposed individuals ineach area were categorized according to their antibody reactivity towarddifferent numbers of salivary P. papatasi proteins. Each value representsthe mean value ± S.E.M. Different letters indicate a significant differencebetween groups (P< 0.05)

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Author's personal copy

inflammation and immunity. Infect Genet Evol 28:691–703. doi:10.1016/j.meegid.2014.07.028

Ahmed SB, Kaabi B, Chelbi I, Derbali M, Cherni S, Laouini D, Zhioua E(2010) Lack of protection of pre-immunization with saliva of long-term colonized Phlebotomus papatasi against experimental chal-lenge with Leishmania major and saliva of wild-caught P. papatasi.Am J TropMed Hyg 83:512–514. doi:10.4269/ajtmh.2010.09-0687

Andrade BB, de Oliveira CI, Brodskyn CI, Barral A, Barral-Netto M(2007) Role of sand fly saliva in human and experimental leishman-iasis: current insights. Scand J Immunol 66:122–127. doi:10.1111/j.1365-3083.2007.01964.x

Barral A, Honda E, Caldas A, Costa J, Vinhas V, Rowton ED, ValenzuelaJG, Charlab R, Barral-Netto M, Ribeiro JM (2000) Human immuneresponse to sand fly salivary gland antigens: a useful epidemiolog-ical marker? Am J Trop Med Hyg 62:740–745

Bates PA (2007) Transmission of Leishmania metacyclic promastigotesby phlebotomine sand flies. Int J Parasitol 37:1097–1106. doi:10.1016/j.ijpara.2007.04.003

Belkaid Y, Kamhawi S, Modi G, Valenzuela J, Noben-Trauth N, RowtonE, Ribeiro J, Sacks DL (1998) Development of a natural model ofcutaneous leishmaniasis: powerful effects of vector saliva and salivapreexposure on the long-term outcome of Leishmania major infec-tion in the mouse ear dermis. J Exp Med 188:1941–1953

Ben Hadj Ahmed S, Chelbi I, Kaabi B, Cherni S, Derbali M, Zhioua E(2010) Differences in the salivary effects of wild-caught versus col-onized Phlebotomus papatasi (Diptera: Psychodidae) on the devel-opment of zoonotic cutaneous leishmaniasis in BALB/cmice. JMedEntomol 47:74–79

Ben Hadj Ahmed S, Kaabi B, Chelbi I, Cherni S, Derbali M, Laouini D,Zhioua E (2011) Colonization of Phlebotomus papatasi changes theeffect of pre-immunization with saliva from lack of protection towardsprotection against experimental challenge with Leishmania major andsaliva. Parasite Vectors 4:126. doi:10.1186/1756-3305-4-126

Carregaro V, Sa-Nunes A, Cunha TM, Grespan R, Oliveira CJ, Lima-Junior DS, Costa DL, Verri WA Jr, Milanezi CM, Pham VM, BrandDD, Valenzuela JG, Silva JS, Ribeiro JM, Cunha FQ (2011)Nucleosides from Phlebotomus papatasi salivary gland amelioratemurine collagen-induced arthritis by impairing dendritic cell func-tions. J Immunol 187:4347–4359. doi:10.4049/jimmunol.1003404

Cecilio P, Perez-Cabezas B, SantaremN,Maciel J, Rodrigues V, Cordeiroda Silva A (2014) Deception and manipulation: the arms of leish-mania, a successful parasite. Front Immunol 5:480. doi:10.3389/fimmu.2014.00480

Chang HK, Thalhofer C, Duerkop BA, Mehling JS, Verma S, Gollob KJ,Almeida R, Wilson ME (2007) Oxidant generation by single infect-ed monocytes after short-term fluorescence labeling of a protozoanparasite. Infect Immun 75:1017–1024. doi:10.1128/iai.00914-06

Charlab R, Valenzuela JG, Rowton ED, Ribeiro JM (1999) Toward anunderstanding of the biochemical and pharmacological complexityof the saliva of a hematophagous sand fly Lutzomyia longipalpis.Proc Natl Acad Sci U S A 96:15155–15160

Chatelain R, Varkila K, Coffman RL (1992) IL-4 induces a Th2 responsein Leishmania major-infected mice. J Immunol 148:1182–1187

Desjeux P (1996) Leishmaniasis. Public health aspects and control. ClinDermatol 14:417–423

Desjeux P (2004) Leishmaniasis: current situation and new perspectives.Comp Immunol Microbiol Infect Dis 27:305–318. doi:10.1016/j.cimid.2004.03.004

Dionisotti S, Zocchi C, Varani K, Borea PA, Ongini E (1992) Effects ofadenosine derivatives on human and rabbit platelet aggregation.Correlation of adenosine receptor affinities and antiaggregatory ac-tivity. Naunyn Schmiedebergs Arch Pharmacol 346:673–676

Elnaiem DE, Meneses C, Slotman M, Lanzaro GC (2005) Genetic vari-ation in the sand fly salivary protein, SP-15, a potential vaccinecandidate against Leishmania major. Insect Mol Biol 14:145–150

Francischetti IM, Valenzuela JG, Pham VM, Garfield MK, Ribeiro JM(2002) Toward a catalog for the transcripts and proteins (sialome)from the salivary gland of the malaria vector Anopheles gambiae. JExp Biol 205:2429–2451

Freshney RI (1987) Culture of animal cells : a manual of basic technique,2nd edn. A.R. Liss, New York

Gantt KR, Goldman TL, McCormick ML, Miller MA, Jeronimo SM,Nascimento ET, Britigan BE, Wilson ME (2001) Oxidative re-sponses of human and murine macrophages during phagocytosisof Leishmania chagasi. J Immunol 167:893–901

Geraci NS, Mukbel RM, KempMT, Wadsworth MN, Lesho E, StaybackGM, Champion MM, Bernard MA, Abo-Shehada M, Coutinho-Abreu IV, Ramalho-Ortigao M, Hanafi HA, Fawaz EY, El-Hossary SS, Wortmann G, Hoel DF, McDowell MA (2014)Profiling of human acquired immunity against the salivary proteinsof Phlebotomus papatasi reveals clusters of differential immunore-activity. Am J Trop Med Hyg 90:923–938. doi:10.4269/ajtmh.13-0130

Gomes RB, Brodskyn C, de Oliveira CI, Costa J, Miranda JC, Caldas A,Valenzuela JG, Barral-Netto M, Barral A (2002) Seroconversionagainst Lutzomyia longipalpis saliva concurrent with the develop-ment of anti-Leishmania chagasi delayed-type hypersensitivity. JInfect Dis 186:1530–1534. doi:10.1086/344733

Herwaldt BL (1999) Leishmaniasis. Lancet 354:1191–1199. doi:10.1016/s0140-6736(98)10178-2

Janini R, Saliba E, Kamhawi S (1995) Species composition of sand fliesand population dynamics of Phlebotomus papatasi (Diptera:Psychodidae) in the southern Jordan Valley, an endemic focus ofcutaneous leishmaniasis. J Med Entomol 32:822–826

Kamhawi S (2000) The biological and immunomodulatory properties ofsand fly saliva and its role in the establishment of Leishmania infec-tions. Microbes Infect 2:1765–1773

Kamhawi S, Arbagi A, Adwan S, Rida M (1993) Environmental manip-ulation in the control of a zoonotic cutaneous leishmaniasis focus.Arch Inst Pasteur Tunis 70:383–390

Kamhawi S, Abdel-Hafez SK, Molyneux DH (1995) A comprehensiveaccount of species composition, distribution and ecology ofphlebotomine sandflies in Jordan. Parasite 2:163–172

Kamhawi S, Belkaid Y, Modi G, Rowton E, Sacks D (2000) Protectionagainst cutaneous leishmaniasis resulting from bites of uninfectedsand flies. Science 290:1351–1354

Katz O, Waitumbi JN, Zer R, Warburg A (2000) Adenosine, AMP, andprotein phosphatase activity in sandfly saliva. Am J Trop Med Hyg62:145–150

Kebaier C, Uzonna JE, Beverley SM, Scott P (2006) Immunization withpersistent attenuated Delta lpg2 Leishmania major parasites requiresadjuvant to provide protective immunity in C57BL/6 mice. InfectImmun 74:777–780. doi:10.1128/iai.74.1.777-780.2006

Kimblin N, Peters N, Debrabant A, Secundino N, Egen J, Lawyer P, FayMP, Kamhawi S, Sacks D (2008) Quantification of the infectiousdose of Leishmania major transmitted to the skin by single sandflies. Proc Natl Acad Sci U S A 105:10125–10130. doi:10.1073/pnas.0802331105

Lane RP, Crosskey RW (1993) British museum (natural history). Medicalinsects and arachnids, 1st edn. Chapman & Hall, London

Lestinova T, Vlkova M, Votypka J, Volf P, Rohousova I (2015)Phlebotomus papatasi exposure cross-protects mice againstLeishmania major co-inoculated with Phlebotomus duboscqi sali-vary gland homogenate. Acta Trop 144:9–18. doi:10.1016/j.actatropica.2015.01.005

Lewis J (1978) The Phlebotomine sanflies (Diptera: Psychodidae) of theoriental region. In: Museum B, Museum NH (eds) Bulletin of theBritish museum (natural history) entomology, vol 37. The Museum,London, pp 217–343

Marzouki S, Ben Ahmed M, Boussoffara T, Abdeladhim M, Ben Aleya-Bouafif N, Namane A, Hamida NB, Ben Salah A, Louzir H (2011)

Parasitol Res

Author's personal copy

Characterization of the antibody response to the saliva ofPhlebotomus papatasi in people living in endemic areas of cutane-ous leishmaniasis. Am J Trop Med Hyg 84:653–661. doi:10.4269/ajtmh.2011.10-0598

Marzouki S, Abdeladhim M, Abdessalem CB, Oliveira F, Ferjani B,Gilmore D, Louzir H, Valenzuela JG, Ben Ahmed M (2012)Salivary antigen SP32 is the immunodominant target of the antibodyresponse to Phlebotomus papatasi bites in humans. PLoS Negl TropDis 6:e1911. doi:10.1371/journal.pntd.0001911

Marzouki S, Kammoun-Rebai W, Bettaieb J, Abdeladhim M, HadjKacem S, Abdelkader R, Gritli S, Chemkhi J, Aslan H, KamhawiS, Ben Salah A, Louzir H, Valenzuela JG, Ben Ahmed M (2015)Validation of recombinant salivary protein PpSP32 as a suitablemarker of human exposure to Phlebotomus papatasi, the vector ofLeishmania major in Tunisia. PLoSNegl Trop Dis 9:e0003991. doi:10.1371/journal.pntd.0003991

Mosleh IM, Geith E, Natsheh L, Abdul-Dayem M, Abotteen N (2008)Cutaneous leishmaniasis in the Jordanian side of the Jordan Valley:severe under-reporting and consequences on public health manage-ment. Trop Med Int Health 13:855–860. doi:10.1111/j.1365-3156.2008.02063.x

Oliveira F, Lawyer PG, Kamhawi S, Valenzuela JG (2008) Immunity todistinct sand fly salivary proteins primes the anti-Leishmania im-mune response towards protection or exacerbation of disease.PLoS Negl Trop Dis 2:e226. doi:10.1371/journal.pntd.0000226

Oliveira F, Rowton E, AslanH, Gomes R, Castrovinci PA, Alvarenga PH,Abdeladhim M, Teixeira C, Meneses C, Kleeman LT, Guimaraes-Costa AB, Rowland TE, Gilmore D, Doumbia S, Reed SG, LawyerPG, Andersen JF, Kamhawi S, Valenzuela JG (2015) A sand flysalivary protein vaccine shows efficacy against vector-transmittedcutaneous leishmaniasis in nonhuman primates. Sci Transl Med 7:290ra90. doi:10.1126/scitranslmed.aaa3043

Ramalho-OrtigaoM, Coutinho-Abreu IV, Balbino VQ, Figueiredo CA Jr,Mukbel R, Dayem H, Hanafi HA, El-Hossary SS, Fawaz Eel D,Abo-Shehada M, Hoel DF, Stayback G, Wadsworth M, ShoueDA, Abrudan J, Lobo NF, Mahon AR, Emrich SJ, Kamhawi S,Collins FH, McDowell MA (2015) Phlebotomus papatasi SP15:mRNA expression variability and amino acid sequence polymor-phisms of field populations. Parasite Vectors 8:298. doi:10.1186/s13071-015-0914-2

Reithinger R, Dujardin JC, Louzir H, Pirmez C, Alexander B, Brooker S(2007) Cutaneous leishmaniasis. Lancet Infect Dis 7:581–596. doi:10.1016/s1473-3099(07)70209-8

Ribeiro JM, Francischetti IM (2003) Role of arthropod saliva in bloodfeeding: sialome and post-sialome perspectives. Annu Rev Entomol48:73–88. doi:10.1146/annurev.ento.48.060402.102812

Ribeiro JM, Katz O, Pannell LK,Waitumbi J,Warburg A (1999) Salivaryglands of the sand fly Phlebotomus papatasi contain

pharmacologically active amounts of adenosine and 5′-AMP. JExp Biol 202:1551–1559

Rogers ME, Ilg T, Nikolaev AV, Ferguson MA, Bates PA (2004)Transmission of cutaneous leishmaniasis by sand flies is enhancedby regurgitation of fPPG. Nature 430:463–467

Rohousova I, Volf P (2006) Sand fly saliva: effects on host immuneresponse and Leishmania transmission. Folia Parasitol (Praha) 53:161–171

Rohousova I, Ozensoy S, Ozbel Y, Volf P (2005) Detection of species-specific antibody response of humans and mice bitten by sand flies.Parasitology 130:493–499

Rohousova I, Hostomska J, Vlkova M, Kobets T, Lipoldova M, Volf P(2011) The protective effect against Leishmania infection conferredby sand fly bites is limited to short-term exposure. Int J Parasitol 41:481–485. doi:10.1016/j.ijpara.2011.01.003

Sacks DL, Kenney RT, Kreutzer RD, Jaffe CL, Gupta AK, Sharma MC,Sinha SP, Neva FA, Saran R (1995) Indian kala-azar caused byLeishmania tropica. Lancet 345:959–961

Schlein Y, Muller GC (2010) Experimental control of Phlebotomuspapatasi by spraying attractive toxic sugar bait (ATSB) on vegeta-tion. Trans R Soc Trop Med Hyg 104:766–771. doi:10.1016/j.trstmh.2010.08.014

Silva F, Gomes R, Prates D, Miranda JC, Andrade B, Barral-Netto M,Barral A (2005) Inflammatory cell infiltration and high antibodyproduction in BALB/c mice caused by natural exposure toLutzomyia longipalpis bites. Am J Trop Med Hyg 72:94–98

Titus RG, Ribeiro JM (1988) Salivary gland lysates from the sand flyLutzomyia longipalpis enhance Leishmania infectivity. Science 239:1306–1308

Titus RG, Bishop JV, Mejia JS (2006) The immunomodulatory factors ofarthropod saliva and the potential for these factors to serve as vac-cine targets to prevent pathogen transmission. Parasite Immunol 28:131–141. doi:10.1111/j.1365-3024.2006.00807.x

Valenzuela JG, Belkaid Y, Garfield MK,Mendez S, Kamhawi S, RowtonED, Sacks DL, Ribeiro JM (2001a) Toward a defined anti-Leishmania vaccine targeting vector antigens: characterization of aprotective salivary protein. J Exp Med 194:331–342

Valenzuela JG, Belkaid Y, Rowton E, Ribeiro JM (2001b) The salivaryapyrase of the blood-sucking sand fly Phlebotomus papatasi be-longs to the novel Cimex family of apyrases. J Exp Biol 204:229–237

Volf P, Tesarova P, Nohynkova EN (2000) Salivary proteins and glyco-proteins in phlebotomine sandflies of various species, sex and age.Med Vet Entomol 14:251–256

WHO (2015) WHO report on global surveillance of epidemic-prone in-fectious diseases - leishmaniasis. WHO. http://www.who.int/csr/resources/publications/CSR_ISR_2000_1leish/en/. Accessed 15April 2015

Parasitol Res

Author's personal copy